Ischemia-reperfusion (IR) injury is a phenomenon in which hypoxic tissue undergoes prolonged damage after the return of oxygenated blood, proving a prevalent clinical challenge faced in organ transplant, and ischemic heart, lung and kidney diseases. Ultimately, IR injury can lead to increased infarct size, organ rejection and organ failure. The purine nucleoside adenosine is produced extracellularly in response to IR injury, and elicits cardioprotective, pulmonary protective and renal protective effects through agonizing adenosine G-protein coupled receptors. However, the half-life of extracellular adenosine is extremely short-lived, as specialized integral membrane transport proteins mediate the rapid membrane permeation of adenosine, where the nucleoside is ultimately metabolized within the cytosol. Human equilibrative nucleoside transporters (hENTs) are the main cellular adenosine transporters. Furthermore, adenosine reuptake inhibitors (AdoRIs), a chemically diverse class of hENT inhibitors, potentiate extracellular adenosine signaling by preventing its rapid reuptake through hENTs. Therefore, select AdoRIs are clinically used as vasoactive agents in the treatment of cardiopathy and renal disorders. However, current AdoRIs are limited in their clinical effectiveness due to their poor pharmacological properties and toxicities. Efforts to improve current AdoRIs or develop novel AdoRIs has been challenged by the lack of atomic-level information on hENTs and the mechanism of AdoRIs. This proposed research seeks to address this gap in knowledge by employing molecular, cellular, and chemical approaches to interrogate features of adenosine reuptake inhibition, adenosine recognition and the transport mechanism exhibited by hENTs. Notably, the rational design of novel adenosine reuptake inhibitors displaying improved subtype specificity will be pursued using cardiac and renal model systems. This work will uncover the molecular features of AdoRI activity, adenosine recognition, along with the transport mechanism exhibited by hENTs. In total, successful completion of this work will provide the framework for improved pharmacological intervention of adenosine biology, which will have far-reaching implications in the treatment of ischemic heart, lung, and kidney disease.